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Abstract

Background

Oncolytic virotherapy for cancer treatment utilizes viruses for selective infection
and death of cancer cells without any adverse effect on normal cells. We previously
reported that the human respiratory syncytial virus (RSV) is a novel oncolytic virus
against androgen-independent PC-3 human prostate cancer cells. The present study extends
the result to androgen-dependent prostate cancer, and explores the underlying mechanism
that triggers RSV-induced oncolysis of prostate cancer cells.

Results

We show that RSV imposes a potent oncolytic effect on LNCaP prostate cancer cells.
RSV infectivity was markedly higher in LNCaP cells compared to the non-tumorigenic
RWPE-1 human prostate cells. The enhanced viral burden led to LNCaP cell apoptosis
and growth inhibition of LNCaP xenograft tumors in nude mice. A functional host immune
response did not interfere with RSV-induced oncolysis, since growth of xenograft tumors
in syngeneic C57BL/6J mice from murine RM1 cells was inhibited upon RSV administration.
LNCaP cells failed to activate the type-I interferon (IFNα/β)-induced transcription
factor STAT-1, which is required for antiviral gene expression, although these cells
could produce IFN in response to RSV infection. The essential role of IFN in restricting
infection was further borne out by our finding that neutralizing IFN activity resulted
in enhanced RSV infection in non-tumorigenic RWPE-1 prostate cells.

Conclusions

We demonstrated that RSV is potentially a useful therapeutic tool in the treatment
of androgen-sensitive and androgen-independent prostate cancer. Moreover, impaired
IFN-mediated antiviral response is the likely cause of higher viral burden and resulting
oncolysis of androgen-sensitive prostate cancer cells.

Background

Oncolytic virotherapy, which takes advantage of selective viral infection and apoptosis
in cancer cells due to robust viral replication, is emerging as an important alternative
to conventional cancer treatment modalities [1-4]. Evidence indicates that concurrent use of a repertoire of different oncolytic viruses
(with different modes of action) may produce more efficacious therapeutic response.
Human respiratory syncytial virus (RSV) is a respiratory tract-specific enveloped
non-segmented negative sense single stranded RNA (NNS) virus of the paramyxovirus
family [5,6]. We have recently identified RSV as an oncolytic virus by demonstrating that RSV
can cause apoptosis of PC-3 human prostate cancer cells in culture and in a xenograft
tumor environment as a consequence of excessive viral replication in PC-3 cells that
led to apoptotic cell death [7]. Specificity of the virus-induced oncolysis of cancer cells was evident from the
lack of significant viral burden and apoptosis of non-tumorigenic human prostate epithelial
cells, such as RWPE-1.

Metastatic prostate cancer is a leading cause of cancer deaths in men in the United
States. The steroid hormone androgen is a potent mitogen for normal and cancerous
prostate epithelial cells. The cognate androgen receptor (AR) mediates nuclear responses
to androgen signaling [8,9]. Although initially androgen-sensitive, metastatic prostate cancer evolves to a state
of androgen independence for growth and proliferation, despite continued expression
of AR at all stages of the disease. AR was shown to activate the transcriptional program
of a distinct set of gene networks, including many genes involved in cell cycle regulation,
during progression of the cancer cells to androgen independence [9]. As noted above, RSV can induce oncolysis of androgen-independent PC-3 prostate cancer
cells and RSV caused regression of PC-3 cell derived xenograft tumors in immune-deficient
nude mice [7]. Extending this study, we examined susceptibility of the androgen-sensitive LNCaP
human prostate cancer cells and LNCaP xenograft tumors to RSV-induced oncolysis, and
the impact of host immune-competence on the oncolytic activity of RSV. Innate immunity
is the first line of antiviral defense for restricting virus growth and spread. Since
both NF-κB and type-I interferon (IFNα/β)-mediated JAK/STAT signaling [10] is required for innate antiviral response; we also examined the activation status
of NF-κB and IFN-induced JAK-STAT pathways in RSV-infected prostate cancer cells.

Herein we report that RSV is potently oncolytic against androgen-sensitive LNCaP human
prostate cancer cells in vitro and in vivo, and aberrant IFN-regulated signaling accounts for LNCaP cell susceptibility to RSV.
While RWPE-1 non-tumorigenic prostate cells were protected against RSV infection by
activation of JAK-STAT and NF-κB signaling, a lack of sustained NF-κB activation was
associated with the susceptibility of PC-3 androgen-independent prostate cancer cells
to RSV-induced oncolysis, although IFN-mediated signaling was functional in these
cells.

RSV (1 MOI) was added to cells for adsorption at 37°C for 1.5 h. After washing, infection
continued for additional 0 h-48 h. At various time points post-infection, virus yields
in culture supernatants were assayed by plaque assay of the monolayer of CV-1 cells
(African green monkey kidney cells) covered with a nutrient medium in methyl-cellulose
[7]. Crystal violet staining of living cells allowed clear visualization of the plaques.
Cell morphology was visualized microscopically. In some experiments, cells were pre-treated
with 1000 units/ml IFN-α for 16 h, followed by infection with RSV for 24 h in the
presence of IFN. Medium supernatants were used to measure viral titer (plaque assay).

4-6-week-old athymic nude male mice (Harlan Laboratories Inc.) were injected subcutaneously
with 3 × 106 LNCaP cells at a site below the ear [7]. When tumors reached palpable size (sizes ranging from 100-300 mm3 for individual mice), RSV (at 1 × 106 pfu per animal) or Opti-MEM (Medium, carrier control) was injected repeatedly into
the tumor mass (intratumoral administration) at 2-day intervals over a two-week period.
Tumor volumes were measured by a caliper (4/3 × 3.14 × r12 × r2 with r1 < r2) and normalized to the tumor volume for the corresponding mouse at day-1 when the
first injection was administered. Tumor samples from euthanized mice were excised
and processed for histology and for TUNEL assay to evaluate apoptosis.

For imaging of subcutaneous xenograft tumors, which were generated using luciferase-expressing
LNCaP-Luc-2 cells, palpable tumors were injected with RSV (1 × 106 pfu per animal via the intratumoral (I.T.) route) or Opti-MEM (Medium, carrier control).
At various days post-infection, luciferin was injected and real-time tumor bioluminescence
(reflecting tumor growth) was monitored in live animals using the Xenogen IVIS imaging
system (Caliper Life Sciences, Hopkinton, MA).

To analyze prostate tumors in syngeneic C57BL/6J mice, 4-6 week-old mice were injected
subcutaneously with RM1 murine prostate cancer cells (1 × 106 cells) in the right dorsal flank. Tumors (tumor volumes ranging from 56-164 mm3 for individual mice) were injected intra-tumorally with either RSV (at 1 × 106 pfu, each animal) or Opti-MEM. The tumor volume for each animal was normalized to
the corresponding volume recorded just prior to the first injection.

Housing and all procedures involving animals were performed according to protocols
approved by the Institutional Committee for Animal Care and Use of the University
of Texas Health Science Center San Antonio.

Histopathology and TUNEL

Part of tumor tissues (excised immediately after euthanasia) was fixed in 10% neutral
buffered formalin, embedded in paraffin, sectioned at 5 micron and analyzed for histology
after staining with hematoxylin and eosin. TUNEL staining in situ was performed using DeadEnd Colorimetric kit (Promega, WI). For each tumor sample,
pathologic changes were graded by a blinded board certified veterinary pathologist
(GBH). Grading criteria were based on invasion of adjacent normal tissue, necrosis,
mitotic figures per 20X high power field, edema, congestion, compression of the surrounding
tissue, mineralization, inflammation (granulocytes, lymphocytes, mixed), hemorrhage,
neovascularization and hemosiderin deposition.

Statistics

Differential tumor growth was determined utilizing STATA Analysis and Statistical
Software (College Station, Texas, USA). Results are mean values ± Standard Error (SE)
of the mean. For each mouse, the tumor volume at every time point was normalized to
its tumor volume at the start of RSV (or Medium) injection, which was set as 100%.
Significance of the difference between Medium- and RSV-injected tumor growth trajectories
was evaluated using linear mixed model that incorporated a random intercept for each
mouse and used normalized mean tumor volume measurements as the dependent variable.
Wald tests assessed statistical significance of differences between growth rates in
two mouse groups.

RSV-induced oncolysis of androgen-dependent and androgen-independent prostate cancer
cells in the absence and presence of androgen

To assess the role of androgen in the RSV effect on LNCaP (androgen-dependent) and
C4-2B (androgen-independent, derived from the parental LNCaP) cells, the cells were
grown in androgen-depleted (charcoal stripped) media and then either ethanol (vehicle)
or R1881a (1 nM) was added to the media for overnight before mock infection or infection
with RSV was performed. RSV replicated efficiently in vehicle-treated and R1881 (1
nM)-treated LNCaP and C4-2B prostate cancer cells (Figure 2a, d). The RSV titer was higher in cells with androgen treatment compared to vehicle treatment.
Cell morphology (Figure 2b, e) revealed that RSV caused oncolysis of LNCaP and C4-2B cells in an androgen-independent
manner.

Figure 2.Oncolytic activity of RSV against androgen-dependent and androgen-independent prostate
cancer cells in the absence and presence of androgen. (a) RSV infection of LNCaP cells (in the presence or absence of androgen) were measured
by plaque assay as described in Figure. 1a. Standard deviations are shown by the error
bars. (b) Morphology of mock-infected or RSV-infected LNCaP cells (in the presence or absence
of androgen). (c) Apoptosis of mock-infected (- RSV) and RSV-infected (+ RSV) LNCaP cells (in the presence
or absence of androgen). Apoptosis assay was performed as described in Figure 1 d.
The figure shows the percentage of cells undergoing apoptosis. Standard deviations
are shown by the error bars. (d) RSV infection of C4-2B cells (in the presence or absence of androgen) were measured
by plaque assay as described in Figure. 1a. Standard deviations are shown by the error
bars. (e) Morphology of mock-infected or RSV-infected C4-2B cells (in the presence or absence
of androgen).

The presence of androgen caused more extensive apoptosis of LNCaP cells (80% apoptosis)
than in the absence of the hormone (50% apoptosis) (Figure 2c). Given the androgen dependence of these cells, the much higher apoptosis of mock-infected
cells in the absence of androgen is expected.

RSV-induced oncolysis of tumors in vivo in mice through apoptosis

Intratumoral administration of RSV to the subcutaneously produced LNCaP xenograft
tumors markedly reduced tumor mass over a three week period, while medium-injected
tumors (carrier control) continued to increase in size over time (Figure 3a). Photomicrographic documentation of tumor regression in a representative mouse is
shown (Figure 3b). Tumor-bearing nude mice receiving medium injection showed drastic reduction in
body weight. This is in contrast to the steady body-weight increase in mice that received
RSV into tumors (Figure 4a). We speculate that the decreased body weight (cancer cachexia) is due to the catabolic
action of the proteolysis-inducing factor (PIF) on skeletal muscle and possibly, to
some extent, also due to the catabolic action of TNF-α (cachectin) on the adipose
tissue and skeletal muscle [16]. Thus, the anti-tumor activity of RSV helps relieve tumor-associated pathophysiological
ailments.

Figure 3.Androgen-sensitive LNCaP xenograft prostate tumor growth in nude mice following intratumoral
injection of RSV. Tumors were produced by subcutaneous injections of LNCaP cells at sites below the
ear. (a) Tumor growth kinetics in mice. Intratumoral injections were given at 2 days apart.
Tumor volume of each mouse was normalized against its tumor volume at day 1 (starting
point) set as 100%. Tumor volumes (normalized) measured on each injection day are
shown in the plot. Each treatment group consisted of four representative animals (n
= 4) and data represent normalized mean tumor volume trajectories over time. Error
bars represent the SE of the mean at each time point. (b) Progressively regressed tumor mass after intratumoral injection of RSV. Tumor growth
corresponding to injections of RSV or Medium was followed. Day 1 represents the initial
injection of RSV or Medium. The Inserts display corresponding tumors extracted at
day-22 before sacrifice.

Figure 4.in vivo oncolytic activity of RSV against androgen-sensitive xenograft prostate tumor. (a) Body weight of tumor bearing mice measured over 14 day following intratumoral administration
of RSV or Medium at 2-day intervals. The body weight data represent the normalized
mean body weight trajectories over time. Body weight of each mouse was normalized
against its body weight at day 1, which was set as 100%. Error bars represent the
SE of the mean at each time point. (b) Real-time bioluminescence imaging of xenograft tumors (generated using luciferase
expressing LNCaP-Luc-2 cells) in live animal following RSV or medium (control) injections
(I.T). Time interval for each injection = 2 days. Whole-body imaging was done with
Xenogen IVIS system. (c) LNCaP xenograft tumor was injected either with medium (control) or RSV (1 × 105 pfu) via I.T. After 3 d post-injection, the tumor was surgically removed and RSV titer
in the tumor homogenate was determined by plaque assay.

Real-time bioluminescence imaging in live mice (using Xenogen IVIS imaging system)
also showed drastic tumor regression by RSV (Figure 4b). The tumor regression was directly due to lytic viral replication in tumor cells,
since high RSV titer was detected in the homogenate of the LNCaP xenograft tumor tissue
that was harvested 3 days after a single RSV injection via I.T (Figure 4c).

Furthermore, the oncolytic effect of RSV is not compromised in the immune-competent
syngeneic host, since RSV significantly inhibited growth of RM1 murine prostate cancer
cell tumors generated in C57BL/6J mice (Figure 5a). We observed that RSV specifically targeted and localized to LNCaP- and RM1-derived
prostate tumors since viral gene specific mRNA expression was detected in tumor lysates,
but not in lysates of various organs (lungs, kidney, liver, spleen) of infected animals
(data not shown). Similar tumor-specific RSV targeting was noted previously in mice
harboring PC-3 derived xenograft tumor [7].

The therapeutic potential of RSV was further evident from its long-term anti-tumorogenic
impact. Xenograft prostate tumors (from luciferase expressing LNCaP-Luc-2 cells),
subjected to four RSV injections (at 2 days apart), resulted in tumor regression and
the tumor failed to reappear even at 44 days after the final RSV injection (Figure
6a). The long-term tumor remission following RSV treatment underscores the feasibility
of developing RSV as an efficient anti-cancer agent.

Figure 6.Sustained tumor remission after RSV administration and subdued immune response to
RSV challenge. (a) RSV was administered (via I. T - four injections at 2-day intervals) to xenograft
tumor (generated from luciferase expressing LNCaP-Luc2 cells). Following 4 injections,
the mouse was observed everyday for a period of 44 days for tumor recurrence. Real-time
bioluminescence imaging of xenograft tumors in the live animal was performed with
Xenogen IVIS system. (b) Levels of interferon-γ (IFNγ) & interleukin-10 (IL-10) in spleen homogenate of normal,
C57BL/6J mice (non-tumor bearing) infected with RSV (4 injections of RSV, 2 days apart
-106 pfu/mouse via i.p.) were measured (by ELISA) at the day-14 following last injection.
Cytokines were also measured in xenograft prostate tumor (subcutaneous tumor generated
using LNCaP cells) 48 hr after RSV injections via I.T. The representative results
are from duplicate experiments with similar values.

Immune responses directed against viruses pose a major hurdle in developing efficient
oncolyitc viruses with potent anti-cancer property. In that regard, immune response
against RSV was minimal, since RSV failed to induce robust immunity following systemic
(via intraperitonial or i.p route) infection of normal, immuno-competent C57BL/6J
mice that did not host xenograft tumors. Very low levels of Th1 (IFN-γ) and Th2 (IL-10)
cytokines (0.25-0.90 pg/ml of IL-10 and IFN-γ respectively, in the spleen homogenate)
in infected animals were indicative of weak immune response following systemic RSV
infection (Figure 6b). Systemic challenge with various foreign agents (bacteria, virus etc) typically
produces 100-3500 pg/ml of IL-10 and IFN-γ in the spleen [17-19].

We also examined whether RSV triggered immune response in the tumor micro-environment.
LNCaP xenograft tumors were injected with RSV (two injections via I.T route; 2-day
apart) and tumor lysate (collected 2 d after the last injection) was analyzed for
the Th1 and Th2 cytokine levels. The almost non-detectable immune mediators (Figure
6b) suggest that tumor regression is not due to host adaptive immunity and self-elimination.
These results along with our data showing lytic RSV replication in the tumors (Figure
4c) suggest that tumor regression occurred mostly due to a direct RSV-mediated apoptosis
of tumor cells.

Tumor pathology

After administering 7 injections of RSV or medium intra-tumorally (injected every
other day), tumors were examined for pathological status. The most prominent change
for RSV-injected tumors, compared to medium-injected tumors, was complete necrosis
with no histologically discernable tumor cells (Figure 7). RSV infected tumors showed markedly reduced size containing a central necrotic
core surrounded by a fibrous pyogranulamatous capsule. The normal inflammatory process
led to significant healing, as revealed by replacement of the tumor mass by non-malignant
tissue (compare Figure 7d with Figure 7a). Histological evaluations of RSV-injected versus medium-injected tumors (3 tumor
specimens in each group) are presented in Table 1. Differences between the control and experimental group are easily discernable. Differences
were most prominent in the degree of invasiveness, (4, 4 and 5 vs. 0, 0 and 0), mitotic
figures per high power field (2, 11 and 22 vs. 0, 0 and 0), necrosis (2, 1 and 2 vs.
5, 5 and 5), compression of adjacent tissue (2, 1 and 2 vs. 0, 0 and 0), mineralization
(0, 0 and 0) vs. 4, 3 and 3). Differences for several additional criteria (edema,
congestion, hemorrhage, and hemosiderin deposition) were not significant.

Loss of IFN-regulated antiviral defense in LNCaP cells

A critical antiviral role of type-I IFN cytokines in restricting infection from RSV
and various other viruses such as measles and vesicular stomatitis virus has been
demonstrated [10,20-23]. Therefore, we investigated whether loss of IFN-mediated antiviral defense mechanism
would account for differential oncolytic activity in LNCaP versus RWPE-1 cells.

Deregulation of IFN-mediated antiviral response could occur due to either insufficient
IFN production from cancer cells, or a dysfunctional IFN-activated JAK/STAT antiviral
pathway. RSV-infected LNCaP cells produced high levels of IFN - even more than RWPE-1
cells (Figure 8a). However, the antiviral activity of IFN (as measured from the viral titer of the
RSV-infected cells) was at least 100-fold higher in the case of RWPE-1 and PC-3 compared
to LNCaP cells (Figure 8b). For these experiments (Figure 8b), cells were pre-treated with IFN for 16 h, followed by RSV infection in the continued
presence of IFN. The viral titer was measured by performing plaque assay of medium
supernatants. Representative plaque assay shows that IFN treatment caused drastic
reduction of RSV infectivity in RWPE-1 (12 h and 24 h post-infection), while failing
to significantly inhibit RSV infectivity/replication in LNCaP cells at 12 h and 24
h post-infection (Figure 8c). In contrast to LNCaP cells, PC-3 cells were protected against RSV to the antiviral
action of IFN, since IFN treatment of PC-3 cells drastically inhibited virus replication
(Figure 8d). In the absence of protection from IFN, RSV has selective growth advantage in LNCaP
cells over RWPE-1 cells and PC-3 cells. Indeed, the IFN neutralizing antibody, which
inhibited IFN-α/β mediated antiviral activity in RWPE-1 cells, caused significant
elevation of the RSV titer in RWPE-1 cells (by approximately 15 fold), representing
enhancement of viral infectivity by 750% (Figure 9a). A representative result from plaque assay of RSV-infected RWPE-1 cells that were
pretreated with either control antibody or IFN-neutralizing antibody shows elevated
viral titer in cells devoid of IFN response (Figure 9b). Results from Figure 9 demonstrate that IFN plays an important role in limiting RSV infection in RWPE-1
and PC-3 cells, whereas lack of this restriction in LNCaP cells associated with excessive
viral replication and oncolysis.

We had reported earlier [7] that PC-3 cells are susceptible to RSV-induced apoptosis despite an intact IFN-responsive
antiviral response (Figure 8b). This is due to a defect in the NF-κB-mediated antiviral response, which was compromised
within 6-12 h post-infection (Figure 10c). However, RSV-infected LNCaP and RWPE-1 cells were not impaired for NF-κB activation
(Figures 10a, b).

Lack of IFN-regulated STAT-1 activation in RSV-infected LNCaP cells

Following engagement with the cognate IFN-receptor, IFN-α/β stimulates a signaling
cascade (JAK/STAT pathway) that culminates in activation of the transcription factors
STAT-1 and STAT-2 (STAT-1/2), which translocates to the nucleus to transactivate antiviral
genes [24]. Although LNCaP cells secreted high levels of IFN (Figure 8a), unlike RWPE-1 and PC-3 cells, RSV-infected LNCaP cells did not activate STAT-1,
revealed from the lack of STAT-1 binding to the cognate DNA element in LNCaP cells
(Figure 11a) in contrast to RWPE-1 (Figure 11a) and PC-3 (Figure 11b) cells, which showed robust STAT-1 activation. EMSA complexes in RWPE-1 and PC-3
cells are shown by the arrow, and specificity of the EMSA is shown by the loss of
STAT-1-specific EMSA complex in competition assay (Figure 11a, b).

We conclude that inability of LNCaP cells to activate STAT-1 in response to RSV infection
is due to a globally dysfunctioning IFN pathway, since IFN treatment did not activate
STAT-1 in LNCaP cells (Figure 11c).

Discussion

Our study has been the first to demonstrate RSV is an oncolytic virus, and this oncolytic
activity is functional in vivo both in immune-deficient nude mice and in an immune-competent host environment, since
RSV inhibited prostate tumor growth in syngeneic C57BL/6J mice. RSV infectivity and
the virus-induced apoptotic index in vitro were much higher in androgen-dependent LNCaP cells compared to non-tumorigenic RWPE-1
prostate cells. Aberrant type I interferon (IFN)-dependent antiviral defense response
[10], which culminated in impaired activation of the STAT-1 transcription factor (STAT-1
is required for expression of IFN-dependent antiviral genes), associated with the
high RSV burden in infected LNCaP cells. We conclude that blockade in STAT-1 activation,
leading to inhibition in the expression of critical IFN-dependent antiviral genes,
accounts for excessive RSV replication leading to apoptosis of LNCaP cells. This is
unlike PC-3 androgen-independent prostate cancer cells for which RSV-induced oncolysis
was associated with failure in a sustained NF-κB activation, which would cause failure
in the induction of NF-κB dependent antiviral genes.

Using IFN-neutralizing antibody, we also provide the first direct evidence (Figure
9) that protection of non-malignant epithelial cells against virus-induced oncolysis
is due to IFN-mediated antiviral defense response. Our results further revealed that
the oncolytic function of RSV may remain active even when the IFN-regulated antiviral
pathway is functional, provided a second defense arm involving NF-κB signaling is
deregulated.

Innate immunity is the first line of defense mounted by the host to combat virus infection
before an orchestrated adaptive immune response is launched [10,20]. IFN-mediated activation of the JAK/STAT antiviral pathway is recognized as a major
antiviral innate immune defense mechanism [24]. In this regard, we have recently demonstrated that RSV-infected lung epithelial
cells and immune cells (e.g. macrophages) utilize Nod2 protein as a molecular sensor
to induce production of IFN-α/β from infected cells after interacting with viral single-stranded
RNA genome and subsequently triggering innate antiviral response [25]. IFN-α/β, which are potent antiviral cytokines produced in infected cells, bind to
cognate cell surface receptors on uninfected cells (via autocrine/paracrine action)
to induce the JAK/STAT antiviral pathway; which helps promote nuclear translocation
and activation of the transcription factors STAT-1 and STAT-2 that in turn activate
antiviral genes [24]. We also reported that an IFN-independent innate defense mechanism involving TNF-α
-induced activation of NF-κB can restrict virus replication in infected cells due
to induction of antiviral genes [15,26]. These two antiviral pathways mediated by IFN (via the JAK/STAT pathway) and TNF-α
(via the NF-κB pathway) are activated in infected cells either individually or together
to coordinate the transcriptional induction of the antiviral gene network.

A large number of cancer cells are deficient in the IFN signaling cascade [27,28], making many types of cancer cells susceptible to apoptosis by oncolytic viruses.
In the context of prostate cancer, our results suggest that both androgen-dependent
prostate cancer cells (such as LNCaP cells) and androgen-independent prostate cancer
cells (such as PC-3 and RM1 cells) are susceptible to RSV-induced oncolysis. We show
that, while IFN production from infected LNCaP cells was normal, IFN failed to activate
STAT-1 in LNCaP cells. In fact, it was reported earlier that LNCaP cells fail to express
JAK1 [29]. On the other hand, RSV infection and IFN treatment of non-tumorogenic RWPE-1 cells
and PC-3 cells was associated with robust STAT-1 activation and protection against
RSV-induced apoptosis. We also show that while PC-3 cells respond to IFN and induce
DNA-binding activity of STAT-1 (in agreement with previous reports that IFN-treated
PC-3 cells are activated for antiviral signaling; [30,31]), impaired NF-κB activation is associated with apoptosis in RSV-infected PC-3 cells.
LNCaP cells, on the other hand, were competent to activate NF-κB in response to RSV
infection. We speculate that androgen dependence and/or the androgen receptor expression
status of prostate cancer cells may influence RSV-mediated modulation of the innate
antiviral apparatus (NF-κB activation vs. IFN-mediated JAK/STAT activation). Our results
(Figures 8, 9, 10, 11) [7] lead us to conclude that deregulation of the IFN pathway in androgen-sensitive LNCaP
prostate cancer cells accounts for loss of STAT-1 activation (and non-expression of
antiviral factors), higher RSV replication, induction of apoptosis and reduced cell
viability, whereas deregulation of the NF-κB-dependent antiviral defense in androgen-independent
PC-3 prostate cancer cells accounts for susceptibility of these cells to RSV-induced
apoptosis.

Advanced-stage cancer cells, which continue to express the androgen receptor in a
majority of tumor specimens, are resistant to apoptosis from androgen ablation or
from the cytotoxicity induced by chemotherapeutic agents. Development of treatment
protocols that would promote prostate cancer cell apoptosis and prevent cancer cell
progression to androgen independence has remained a major challenge in prostate cancer
therapy. To this end, it is tempting to speculate that complete ablation of prostate
cancer cells at an early stage, when the cells are still androgen-sensitive, is likely
to prevent clonal emergence of androgen-independent prostate cancer cells. The anti-tumor,
oncolytic activity of RSV against androgen receptor-negative prostate tumors has additional
clinical significance, since reduced or non-detectable androgen receptor expression
has been observed in a small percent of metastatic neoplastic foci at distant organ
sites from castrate resistant prostate cancer patients [32,33]. The observation that RSV-induced oncolysis of prostate tumors can occur in immune-competent
C57BL/6J mice has obvious clinical significance. It is important to mention that systemic
delivery of RSV represent a clinically feasible route for therapy. In that regard,
we have previously demonstrated that intraperitoneal (i.p.) injections of RSV are
effective in causing regression of PC-3 xenograft tumors [7] which are more aggressive than LNCaP xenograft tumors with regard to tumor growth.
Thus, studies are underway to examine oncolytic efficacy of RSV against LNCaP tumors
following systemic administration. The results from our study suggest that RSV-based
therapy has the potential to be a viable strategy for prostate cancer treatment.

Conclusions

In summary, our study demonstrated the oncolytic activity of RSV promotes selective
apoptosis of androgen-sensitive and androgen-refractory prostate cancer cells by exploiting
the deficiency in the antiviral signaling propagated by either the IFN-mediated JAK/STAT
activation or NF-κB activation in virus infected cells. The host immune response did
not interfere with the oncolytic activity of RSV. On the basis of these results we
suggest that the oncolytic property of RSV may be useful in the development of virotherapy
for noncurative, metastatic prostate cancer.

Competing interests

The authors declare that they have no competing interests.

Authors' contributions

All authors have read and approved the final manuscript. The authors made the following
contributions to this work. IE responsible for the study design, experimental work,
data evaluation and analysis and helped write the manuscript. TH was involved in experimental
design, experimental work and critique. AS performed the interferon assays and viral
titer assays. IB was involved in the animal experiments. YK and GBH performed the
histology analysis. BC and SB were the research supervisors and participated in the
study design, assessment of the results, and writing the manuscript.

Acknowledgements

We would like to thank Jesus Segovia for technical assistance. This work was supported
in part by the American Lung Association National Biomedical Research grant (RG-49629-N)
(S. Bose), NIH grants AI069062 (SB), AI083387 (SB), CA129246 (SB; BC), AG10486 & AG19660
(BC), UTHSCSA-UTSA SALSI Grant (SB; BC), VA Merit Review Grant (BC). The work was
also supported by Translational Technology Resources Award (to IE) from UTHSCSA IIMS.
AS was supported by NIH NIDCR grant DE14318 for the COSTAR program. BC is a VA Senior
Research Career Scientist. FACS at the UTHSCSA Core Facility was supported by the
Cancer Center Program grant CA54174 (NIH-P30). Financial assistance was also provided
to SB and BC by the Cancer Therapy and Research Center at the University of Texas
Health Science Center-San Antonio through the NCI Cancer Center Support Grant (2 P30
CA054174-17).